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Genome Editing, DNA double-strand break Repair and cellular Responses (GE2R)

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Genome Editing Tools: From Gene targeting to the study of Genome instabiliy processes

People involved

  • Carine Giovannangeli, DR CNRS
  • Jean-Paul Concordet, CR Inserm
  • Danièle Praseuth, MC Museum
  • Anne De Cian, IR Inserm
  • Loïc Perrouault, IE Museum
  • Charlotte Boix, AI Inserm
  • Alice Brion, IE-MNHN
  • Khadija Lamribet, IE-INSERM
  • Armêl Millet, PhD student*
  • Ahmed Kheder, PhD student
  • Sylvain Geny, Post-Doc

Former Team Member

  • Erika Brunet, CR Inserm*
  • Loelia Babin, PhD Student*
  • Sonia Dubois, Post-Doc
  • Jean-Baptiste-Renaud, IE-CDD
  • Marine Charpentier, PhD Student

Development and optimization of genome editing approaches

Here we are focusing on developing new strategies to optimize genome editing methods using TALE proteins and CRISPR/cas9 system and provide resources in the field (TACGENE Facility). We are characterizing the DNA repair mechanisms associated with nuclease driven genome modifications in order to improve the efficiency of the strategies. Our work includes the biophysical characterization of these sequence-specific DNA agents.


Biochemical and proteomic characterization of DNA repair complexes

We have developed a strategy to directly purify native nucleo-protein complexes and to identify the protein partners involved in these complexes, by mass spectrometry analyses. Thus we can identify new DNA repair proteins and complexes, and cellular activities are validated by using well established DNA repair reporters.


Molecular mechanisms driven chromosomal translocations in human cells and their role in oncogenesis*

This project focuses on understanding chromosomal translocation formation that can arise from two simultaneous DSBs on two heterologous chromosomes. We use site-specific nucleases such as ZFNs, TALENs or CRISPR/Cas9 to induce chromosomal translocations in human cells. The nucleases are directed toward genomic regions implicated in oncogenic translocations. Creating physiologically relevant translocations at endogenous loci, rather than ectopically provides us a physiological model to study translocation mechanisms and to understand the tumorigenesis associated with such chromosomal rearrangements.


Main collaborations

  • Filippo Del Bene, Institut Curie, Paris (Zebrafish gene targeting)
  • Ignacio Anegon, UMR1064, Nantes (Rat transgenesis)
  • Maria Jasin, MKSCC, New-York, USA (DNA repair)
  • Agnel Sfeir, NYU, New York, USA (DNA repair)
  • Bernard Lopez, IGR, Villejuif (DNA repair)
  • Tsuyoshi Momose, CNRS, Villefranche Sur Mer (Clytia gene targeting)
  • Arnaud Poterszman, IGBMC, Strasbourg (Structural Biology)
  • Filippo Rusconi, Univ. Paris-Sud, Orsay (radiation chemistry)
  • Malcolm Buckle, ENS Cachan (SPR imaging)
  • Olivier Piétrement, IGR (AFM)

Selected references

  1. Momose T. & Concordet J-P. (2016) Diving into marine genomics with CRISPR/Cas9 systems. Mar Genomics. pii: S1874-7787(16)30122-2.
  2. Haeussler M., Schönig K., Eckert H., Eschstruth A., Mianné J., Renaud J-B., Schneider-Maunoury S., Shkumatava A., Teboul L., Kent J., Joly J-S. & Concordet J-P. (2016) Evaluation of off-target and on-target scoring algorithms and integration into the guide RNA selection tool CRISPOR. Genome Biol. 17(1):148.
  3. Haeussler M. & Concordet J-P. (2016) Genome Editing with CRISPR-Cas9: Can It Get Any Better? J. Genet. Genomics. 43,5:239-50.
  4. Di Donato V., De Santis F., Auer T-O., Testa N., Sánchez-Iranzo H., Mercader N., Concordet J-P. & Del Bene F. (2016) 2C-Cas9: a versatile tool for clonal analysis of gene function. Genome Res. 26,5:681-92.
  5. Renaud J-B., Boix C., Charpentier M., De Cian A., Cochennec J., Duvernois-Berthet E., Perrouault L., Tesson L., Edouard J., Thinard R., Cherifi Y., Menoret S., Fontanière S., de Crozé N., Fraichard A., Sohm F., Anegon I., Concordet J-P. & Giovannangeli C. (2016) Improved Genome Editing Efficiency and Flexibility Using Modified Oligonucleotides with TALEN and CRISPR-Cas9 Nucleases. Cell Reports. 14,9:2263-72.
  6. Berthelot V., Mouta-Cardoso G., Hégarat N., Guillonneau F., François J-C., Giovannangeli C., Praseuth D. & Rusconi F. (2016) The human DNA ends proteome uncovers an unexpected entanglement of functional pathways. Nucleic Acids Res. 44,10:4721-33.
  7. Piganeau, M., Renouf, B., Ghezraoui, H. & Brunet, E. (2016) TALEN-Induced Translocations in Human Cells. Methods Mol Biol. 1338:99-117.
  8. Tesson, L., Remy, S., Ménoret, S., Usal, C., Thinard, R., Savignard, C., De Cian, A., Giovannangeli, C., Concordet, J-P. & Anegon, I. (2016)  Genome Editing in Rats Using TALE Nucleases. Methods Mol Biol. 1338:245-59.
  9. Ménoret, S., De Cian, A., Tesson, L., Remy, S., Usal, C., Boulé, J-B., Boix, C., Fontanière, S., Crénéguy, A., Nguyen, T-H., Brusselle, L., Thinard, R., Gauguier, D., Concordet, J-P., Cherifi, Y., Fraichard, A., Giovannangeli, C. & Anegon, I.  (2015) Homology-directed repair in rodent zygotes using Cas9 and TALEN engineered proteins. Sci Rep. 7(5):14410. 
  10. Auer, T-O., Duroure, K., Concordet, J-P. & Del Bene, F. (2014) CRISPR/Cas9-mediated conversion of eGFP- into Gal4-transgenic lines in zebrafish. Nat Protoc. 9,12: 2823-40.
  11. Larcher, T., Lafoux, A., Tesson, L., Remy, S., Thepenier, V., François, V., Le Guiner, C., Goubin, H., Dutilleul, M., Guigand, L., Toumaniantz, G., De Cian, A., Boix, C., Renaud, J-B., Cherel, Y., Giovannangeli, C., Concordet, J-P., Anegon, I. & Huchet, C. (2014) Characterization of dystrophin deficient rats: a new model for Duchenne muscular dystrophy. PLoS One. 9,10: e110371.
  12. Renouf, B., Piganeau, M., Ghezraoui, H., Jasin, M. & Brunet, E. (2014) Creating cancer translocations in human cells using Cas9 DSBs and nCas9 paired nicks.Methods Enzymol. 546: 251-71.
  13. Giovannangeli, C. & Concordet, J-P. (2014) Editing and investigating genomes with TALE and CRISPR/Cas systems: applications of artificial TALE and CRISPR-Cas systems. Methods. 69,2: 119-20.
  14. Ghezraoui, H., Piganeau, M., Renouf, B., Renaud, J-B., Sallmyr, A., Ruis, B., Oh, S., Tomkinson, A-E., Hendrickson, E-A., Giovannangeli, C., Jasin, M. & Brunet, E. (2014) Chromosomal translocations in human cells are generated by canonical nonhomologous end-joining. Mol Cell. 55, 6: 829-42.
  15. Rémy, S., Tesson, L., Ménoret, S., Usal, C., De Cian, A., Thepenier, V., Thinard, R., Baron, D., Charpentier, M., Renaud, J-B., Buelow, R., Cost, G-J., Giovannangeli, C., Fraichard, A., Concordet, J-P. & Anegon, I. (2014) Efficient gene targeting by homology-directed repair in rat zygotes using TALE nucleases. Genome Res. 24,8: 1371-83.
  16. Ménoret, S., Tesson, L., Rémy, S., Usal, C., Thépenier, V., Thinard, R., Ouisse, L-H., De Cian, A., Giovannangeli, C., Concordet, J-P. & Anegon, I. (2014). Gene targeting in rats using transcription activator-like effector nucleases. Methods. 69,1: 102-7.
  17. Auer TO, Duroure K, De Cian A, Concordet JP & Del Bene F. (2014). Highly efficient CRISPR/Cas9-mediated knock-in in zebrafish by homology-independent DNA repair. Genome Res, 24, 1:142-53.
  18. Piganeau M, Ghezraoui H, De Cian A, Guittat L, Tomishima M, Perrouault L, René O, Katibah GE, Zhang L, Holmes MC, Doyon Y, Concordet JP, Giovannangeli C, Jasin M & Brunet E. (2013). Cancer translocations in human cells induced by zinc finger and TALE nucleases. Genome Res, 23,7 :1182-93.
  19. Concordet, J. P. & Giovannangeli, C. (2011). Engineered Nucleases for Targeted Genome Modification. Curr Gene Ther, 11, 1: 1.
  20. Nakanishi, K., Cavallo, F., Perrouault, L., Giovannangeli, C., Moynahan, M. E., Barchi, M., Brunet, E. & Jasin, M. (2011). Homology-Directed Fanconi Anemia Pathway Cross-Link Repair Is Dependent on DNA Replication. Nat Struct Mol Biol, 18, 4: 500-3.
  21. Simsek*, D., Brunet*, E., Wong, S. Y., Katyal, S., Gao, Y., McKinnon, P. J., Lou, J., Zhang, L., Li, J., Rebar, E. J., Gregory, P. D., Holmes, M. C. & Jasin, M. (2011). DNA Ligase III Promotes Alternative Nonhomologous End-Joining During Chromosomal Translocation Formation. PLoS Genet, 7, 6: e1002080

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